1. Field of the Invention
The present invention relates generally to the fields of medicine, molecular biology, immunology, and gene therapy. More particularly, it concerns CD19 chimeric-antigen receptor (CAR)-targeting and universal CAR-targeting monoclonal antibodies and methods of using the same.
2. Description of Related Art
Genetically modified T cells engineered to express a tumor-specific chimeric antigen receptor (CAR) have been infused initially with modest (Till et al., 2008; Pule et al., 2008; Morgan et al., 2010) and recently significant anti-tumor effects (Kochenderfer et al., 2010; Kalos et al., 2011; Brentjens et al., 2011; Kochenderfer et al., 2012; O'Connor et al., 2012). The prototypical CAR uses an extracellular domain to directly dock to a cell surface molecule, which is usually a tumor-associated antigen (TAA). The specificity of a CAR is typically derived from a scFv region assembled from the antigen-binding region of a TAA-specific monoclonal antibody (mAb). The components of a second generation CD19-specific CAR currently in use in clinical trials, designated CD19RCD28, are shown in FIG. 1A. The scFv from mouse mAb clone FMC63 (Nicholson et al., 1997) is fused in frame to an extracellular scaffold (e.g., human immunoglobulin hinge and Fc regions or hinge and constant regions from human CD8α) to promote oligomerization after binding TAA, which contributes to CAR-dependent activation via one or more signaling motifs in the endodomain.
Investigators measure the persistence of adoptively transferred T cells in vivo to assess the therapeutic potential of CAR+ T cells (Kalos et al., 2011; June et al., 2009). The most common approach to assessing survival of infused CAR+ T cells is quantitative PCR using CAR-specific primers (Kochenderfer et al., 2010; Morgan et al., 2006). However, this technique does not allow retrieval of genetically modified T cells from the recipient for multi-parameter analyses. What is needed, and is provided herein, is a mAb to specifically detect and isolate CD19-specific CAR+ genetically modified T cells. In addition to informing on persistence, a CAR-specific mAb can be used for in-process testing during manufacture to assess expression of the transgene, such as after electro-transfer of Sleeping Beauty (SB) or piggyBac DNA plasmids coding for CD19RCD28 (Singh et al., 2008; Manuri et al., 2009; Hackett et al., 2010; Singh et al., 2011).
Furthermore, two different reagents for the detection of CAR-modified T cells are known. Brentjens et al. (2013) describes the use of a biotinylated goat anti-mouse IgG (Fab′)2 (Jackson ImmunoReseach) for detection of CAR-modified T cells. As the sensitivity of this polyclonal antibody is low, in the setting of low lymphocyte numbers in patient samples, detection of CAR-modified T cells was accomplished only after non-specific expansion of T cells using Dynabeads. This prevents a direct assessment of circulating CAR-modified T cell in vivo after infusion. Zheng et al. (2012) describes the use of Protein L for detection of CAR-modified T cells by flow cytometry. This reagent has restricted use in terms of detection and sensitivity in a multi-parameter flow cytometry assay. Its use in other assay formats has not been shown. Importantly, both of these reagents are used for detection purposes only and their functionality has not been shown. Thus, there remains a need for reagents that specifically target CAR expressing cells for analysis and manipulation of the cell populations.